Iron is an essential element but is toxic in excess. The concentration of free iron within cells is determined by regulated transmembrane iron import and storage. Malregulation of iron transport results in tissue injury, either from iron deprivation or overload. We propose to identify and characterize genes and proteins that affect the storage of iron in the yeast vacuole and in mammalian lysosomes and determine the mechanisms of high iron toxicity. We will continue our approach of using yeast genetics to identify genes involved in iron metabolism. We have shown that high iron induces a response that up-regulates the expression of the vacuole iron importer Ccc1, which protects yeast cells from iron toxicity. This transcriptional response is regulated by the iron sensitive transcription factor Yap5. We have discovered that transcription of CCC1 is increased by low glucose and iron in a Yap5-independent manner. We will identify the molecules responsible for the Yap5-independent induction of CCC1 and how low glucose affects iron storage. Yeast and mammals also export iron from the vacuole and lysosomes during iron limitation. We will determine if the yeast vacuolar iron exporters, Fet5/Fth1 and Smf3, are regulated post-translationally. Using stringent genetic selection systems, we will identify structural features on transporters that define metal specificity. Vertebrates store iron in ferritin and stored iron can be released from ferritin. Ferrtin can be degraded in the cytosol or in lysosomes, which are the equivalent of yeast vacuoles. We will determine how iron is exported from lysosomes. Iron is stored in ferritin as Fe3+ and must be reduced to Fe2+ to be exported by transporters. We will identify the mechanism of iron reduction and if there is a pool of lysosomal Fe2+. We will determine the role of putative lysosomal iron exporters and if those exporters are regulated at the level of localization, protein stability or activity. Our studies in yeast suggest that iron toxicity can occur both in the presene and absence of oxygen. We will determine the mechanism of oxygen independent iron toxicity. We identified two unique metabolites whose levels increase under conditions of iron toxicity. Through the use of metabolic profiling, we identified two unique metabolites that are only generated by toxic concentrations of iron. We will test the hypothesis that these metabolites are generated by an enzyme that is either mismetallated or altered by high iron. We will also determine if iron-dependent alteration of mitochondrial DNA is a prerequisite for oxygen-dependent iron toxicity. We have shown that alteration of citrate levels exacerbates iron toxicity in yeast. We will determine if alteration of citrate levels affects mammalian iron toxicity.